CN104430306A - Gesneriaceae plant cryopreservation method - Google Patents

Abstract

The invention provides a method for ultra-low temperature preservation of Gesneriaceae plants. The leaf disc method is used to induce adventitious buds, and then the micro-drop vitrification method is used for ultra-low temperature preservation. Chirita dielsii, Chirita speciosa, Lysionotus pauciflorus vac. Pauciflorus, Lysionotus serratus var. Leaf discs of Lysionotus sulphureoides induced adventitious buds and were precultured, loaded and treated with plant vitrification solution. The cryopreserved leaf discs can regenerate whole plants after recovery culture. The cryopreservation methods of the round-leaved chrysanthemum, beautiful chrysanthemum, chrysanthemum, dentate chrysanthemum and Baoshan chrysanthemum chrysanthemum provided by the invention are simple, easy, stable and reliable, and can regenerate leaf disks after ultra-low temperature preservation The plants grew in good condition.

Description

Cryogenic preservation method of Gesneriaceae plants

Technical field:

The invention belongs to the technical field of ultra-low temperature preservation of plant germplasm resources, and in particular relates to a method for ultra-low temperature preservation and a recultivation method of Gesneriaceae plants.

Background technique:

Gesneriaceae has about 133 genera and 3000 species in the world. Widely distributed in tropical and subtropical regions of Africa, Central and South America, East Asia, South Asia, Southern Europe and Oceania. There are 56 genera and about 442 species of Gesneriaceae in China, of which 25 genera and 354 species are endemic to China.

The distribution of Gesneriaceae in China is narrow, the habitat is hidden and the flowering period is inconsistent, and most species are difficult to collect (Gesneriaceae plants in China). The seeds of Gesneriaceae are tiny, and some species are difficult to set (Wen and Qun). The number of some endangered species is very small, and the collection and preservation of their seeds are more difficult. Some species of Gesneriaceae have high requirements on environmental conditions, and the suitable temperature range, humidity range and soil pH range are relatively small. In the process of introduction and cultivation in the northern region, it is difficult for some species to survive. Due to the narrow area of distribution, the concealment of habitat and the inconsistency of flowering period, it is difficult to collect most species. Among the Gesneriaceae plants in China, about 25% of the genera and 30% of the species were collected and named after the 1950s.例如，报春苣苔属在2010年以后被描述的种就至少包括：Primulina mabaensis、Primulina chizhouensis、Primulina debaoensis、Primulinaguangxiensis、Primulina bullata、Primulina huaijiensis、Primulina qingyuanensis、Primulinabeiliuensis、Primulina purpurea、Primulina yangshuoensis、Primulina fengshanensis、 Primulina lutvittata, Primulina sinovietnamica and Primulina cardamifolia. However, the new species of Chirita genus described after 2010 include at least: Chirita grandibracteata, Chirita lutea, Chiritaluochengensis and Chirita luzhaiensis. The above-mentioned recently discovered and described species, regardless of their taxonomic status, represent some special traits, which are important breeding resources for the horticultural industry in constant pursuit of novelty.

Research on cryopreservation of Gesneriaceae plants is limited. In 2004, Moges et al. established a cryopreservation system for African violets (Saintpaulia ionantha Wendl) by embedding dehydration, vitrification, and embedding vitrification using isolated shoot tips as explants. In embedding dehydration, pre-incubation with 0.3M sucrose for 2 days, followed by air dehydration or silica gel dehydration, can obtain regeneration rates of 75% and 30%, respectively. Different osmoprotection in the vitrification process will affect the regeneration rate, and the classic osmoprotection solution works best, and a regeneration rate of 55% can be obtained. In the embedding and dehydration method, the survival rate and regeneration rate can reach 80% respectively. Li Jia used tobacco leaf explants of Chirita heterotricha Merr. and medicinal Chirita medica D.Fang ex W.T.Wang as materials, after natural drying, loading liquid treatment, and vitrification solution treatment , Liquid nitrogen cryopreservation, successfully achieved vitrification ultra-low temperature cryopreservation, and the materials after liquid nitrogen cryopreservation can continue to differentiate and grow. The survival rates reached 50.0% and 27.8%, respectively. Tang Zhenghui used the detached leaf discs as explants to establish the cryopreservation method for the vitrification method of tobacco leaf, lip and hemicapsula. Explants subcultured for 20 days were desiccated at room temperature for 3 days to induce desiccation tolerance, followed by cryopreservation using a classic vitrification cryopreservation procedure. Under optimized conditions, the survival rates of explants after cryopreservation can reach 50.0%, 38.9% and 28.9%, respectively, but only 1-2 buds of the surviving explants can continue to differentiate and grow. At the same time, it was pointed out that the regeneration rate of leaf explants was very low after cryopreservation when adding vitrification procedure after adventitious bud formation. However, the liquid nitrogen freezing time in the experiment was 6 hours, and the explants after 8 hours of freezing did not continue to survive after thawing.

Invention content:

The object of the present invention is to provide an efficient cryopreservation method for Gesneriaceae plants using leaf discs to address the above-mentioned shortcomings in the prior art. The method is simple and easy to implement, stable and reliable, and the Gesneriaceae plants recover and grow well after preservation, and the regeneration rate of the plants is greatly improved.

In order to realize the above-mentioned purpose of the present invention, the present invention provides following technical scheme:

A method for ultra-low temperature preservation of Gesneriaceae plants, the method comprising the steps of:

(1) Take Gesneriaceae plant leaf discs under aseptic conditions, and transfer the leaf discs to adventitious bud induction medium to complete adventitious bud induction;

(2) Carry out pre-culture to the leaf disk that completes adventitious bud induction;

(3) Take the leaf discs that have completed step (2), transfer them to a freezing tube filled with loading liquid, and load them at 25°C for 20 minutes;

(4) The leaf disc in step (3) is placed in the plant vitrification solution, and frozen;

(5) The leaf discs after the treatment in step (4) are put into liquid nitrogen for storage.

According to the preservation method, wherein the Gesneriaceae plant in the step (1) is the Gesneriaceae plants L. chinensis, beautiful L. syringae, hanging stones, tooth leaf hanging stones, Baoshan The leaves of the tissue-cultured seedlings of the above-mentioned plants were taken, and punched into 3mm leaf disks with a puncher to induce adventitious buds.

According to the preservation method, wherein the pre-cultivation in the step (2) is to culture the adventitious buds in MS liquid medium containing 0.3M sucrose in dark at 25° C. for 24 hours.

According to the preservation method, wherein the loading solution in the step (3) is MS basal medium supplemented with 2M glycerol and 0.4M sucrose.

According to the preservation method, wherein the plant vitrification solution in the step (4) is a PVS3 solution, and 50% (w/v) glycerol and 50% (w/v) sucrose are added from the MS basal medium.

According to the preservation method, the treatment time of the plant vitrification solution in the step (4) is as follows: round-leaved lipophyte, beautiful lipophyte, hanging stone lettuce, tooth leaf hanging stone lettuce, Baoshan The hanging stone lettuce was treated with PVS3 for 40, 40, 60, 80, 80 minutes respectively.

According to the preservation method, wherein the step (3) is: transferring the leaf discs to 25°C freezing tubes filled with plant vitrification solution, and each tube is equipped with 10 leaf discs.

According to the preservation method, wherein the freezing treatment in the step (4) is to first transfer the leaf disk onto a strip of sterile aluminum foil, and then directly insert the strip of aluminum foil into liquid nitrogen.

According to the preservation method, the Gesneriaceae plants preserved in step (5) are re-cultivated, and the leaf discs of the Gesneriaceae plants preserved at ultra-low temperature are thawed, and then cultured again, the thawed To remove the aluminum foil strip from the liquid nitrogen, quickly insert it into the unloading solution containing 10ml MS basal medium plus 1.2M sucrose in the Petri dish, and treat it at 25°C for 20 minutes.

According to the preservation method, wherein the recovery culture is to transfer the thawed leaf discs into MS basal medium and add 0.09M sucrose, 0.5mg l ^-1 BA and 2.5gl ^-1 Phytagel to the recovery medium for cultivation , first cultured at 25°C in the dark for 7 days, and then cultured at 25°C with a photoperiod of light/dark 14/10 hours and a light intensity of 40 μmol m ^-2 s ^-1 .

The technical solution provided by the invention realizes the ultra-low temperature preservation of five kinds of Gesneriaceae plants. The method is simple, stable and reliable. After preservation, the five kinds of Gesneriaceae plants recover and grow in good condition, and the plant regeneration rate is greatly improved.

Description of drawings

Fig. 1. Regeneration of shoot tip and leaf discs of C. rotundum in cryopreservation. (a1) A single plant regenerated from the shoot tip in cryopreservation (the scale bar is 1mm). (a2) A cluster of plants regenerated from the shoot tips stored in cryopreservation (the scale bar is 1mm). (b1) A single plant was regenerated from cryopreserved leaf discs (the scale bar is 1mm). (b2) A cluster of plants was regenerated from cryopreserved leaf discs (the scale bar is 1 mm). (c1) Transplanted 3-week-old ultra-low temperature regenerated plants of Lettuce rotundum (the scale is 1 cm). (c2) Plants regenerated from ultra-low temperature bloomed one month after transplanting (the scale is 1 cm).

Fig. 2. Regeneration of cryopreserved leaf disks of Lettuce japonica. (a, b) The regeneration status of cryopreserved leaf discs after thawing for 6 weeks (scale bar is 1mm). (c) The tissue culture line of C. labiata re-established by cryopreserving leaf disks (the scale is 1 cm). (d) Plants regenerated from cryopreserved leaf disks were transplanted into the greenhouse (the scale is 2 cm).

Fig. 3. Regenerated plants after cryopreservation of P. japonica. (a) Tissue culture system re-established by cryopreserving leaf discs (scale bar is 1 cm). (b) Cryopreservation of regenerated plants 3 months after transplantation (the scale is 2 cm). (c) The regenerated plants were cryopreserved 7 months after transplantation (the scale is 2 cm).

Fig. 4. Regeneration of leaf disks preserved in cryopreservation of Lithospermia serrata. (a) Leaf discs treated with cryopreservation but not regenerated (bars are 1 mm). (b) Plants regenerated after cryopreserved leaf discs were recovered for 8 weeks (scale bar is 2 mm). (c) Tissue culture line of Baoshan Diaoshi Lettuce re-established by cryopreserved leaf discs (the scale is 1cm). (d) Plants regenerated from cryopreserved leaf disks were transplanted into the greenhouse (the scale is 1 cm).

Fig. 5. Regeneration of leaf discs preserved in cryopreservation of Baoshan Dianshi Lettuce. (a,b) The cryopreserved leaf discs regenerated after 8 weeks of culture (scale bar is 2mm). (c) Tissue culture line of Baoshan Diaoshi Lettuce re-established by cryopreserved leaf discs (the scale is 1cm). (d) Plants regenerated from cryopreserved leaf disks were transplanted into the greenhouse (the scale is 2 cm).

Fig. 6. The effect of PVS2 treatment time on the regeneration rate of the shoot tips of Cinnamonia rotundum in cryopreservation. After pre-culture and osmotic protection, shoot tips were treated with PVS2 at 0°C for a certain period of time. Freezing and thawing were performed following the droplet vitrification procedure after PVS2 treatment.

Fig. 7. The effect of PVS3 treatment time on the regeneration rate of the shoot tip of Cinnamon rotundus in cryopreservation. Shoot tips were treated with PVS3 at 25°C for a certain period of time after pre-culture and osmotic protection. Freezing and thawing were performed following the droplet vitrification procedure after PVS3 treatment.

Fig. 8. The effect of PVS3 treatment time on the regeneration rate of leaf discs preserved in cryopreservation of circular leaf column. After preculture and osmotic protection, leaf disks were treated with PVS3 at 25°C for a certain period of time. Freezing and thawing were performed following the droplet vitrification procedure after PVS3 treatment.

Figure 9. The effect of PVS3 treatment time on the regeneration rate of cryopreserved leaf disks of P. After preculture and osmotic protection, leaf disks were treated with PVS3 at 25°C for a certain period of time. Freezing and thawing were performed following the droplet vitrification procedure after PVS3 treatment.

Figure 10. The effect of PVS3 treatment time on the regeneration rate of leaf disks preserved in ultra-low temperature preservation of D. After preculture and osmotic protection, leaf disks were treated with PVS3 at 25°C for a certain period of time. Freezing and thawing were performed following the droplet vitrification procedure after PVS3 treatment.

Fig. 11. The effect of PVS3 treatment time on the regeneration rate of leaf disks preserved in ultra-low temperature preservation of Lithospermia serrata. After preculture and osmotic protection, leaf disks were treated with PVS3 at 25°C for a certain period of time. Freezing and thawing were performed following the droplet vitrification procedure after PVS3 treatment.

Figure 12. The effect of PVS3 treatment time on the regeneration rate of leaf disks preserved in ultra-low temperature preservation of Baoshan hanging stone lettuce. After preculture and osmotic protection, leaf disks were treated with PVS3 at 25°C for a certain period of time. Freezing and thawing were performed following the droplet vitrification procedure after PVS3 treatment.

Detailed ways:

The substantive content of the present invention will be further described with the embodiments of the present invention below in conjunction with the accompanying drawings, but it should not be construed as a limitation of the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention shall fall within the scope of the present invention.

Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art.

Technical scheme of the present invention generally comprises the steps:

(1) Use a puncher to obtain discs of 3 mm in diameter under aseptic conditions, such as round-leaf stalks, beautiful stalks, hanging stones, toothed leaves, and Baoshan stalks . The leaf discs were transferred to adventitious bud induction medium to complete adventitious bud induction.

(2) Pre-cultivate the leaf discs after adventitious bud induction.

(3) Take the leaf discs that have completed step (2), transfer them into a freezing tube filled with a loading solution, and perform loading treatment at 25° C. for 20 minutes.

(4) The leaf discs in step (3) are placed in a freezing tube filled with plant vitrification solution, and processed on ice for a certain period of time.

(5) The leaf discs processed in step (4) were transferred into sterile aluminum foil sheets and then directly put into liquid nitrogen. After the cooling process was completed, they were transferred into cryopreservation tubes for ultra-low temperature storage.

In the above method, the step (1) is punching the leaves of the tissue-cultured seedlings of five kinds of Gesneriaceae plants into 3mm leaf disks with a puncher and inducing adventitious buds. The step (2) is to pre-cultivate the leaf discs that have been induced by adventitious buds. The pre-cultivation treatment can reduce the water content of cells, increase the soluble sugar content of cells, improve the dehydration tolerance, thereby improve the freezing resistance of shoot tips, reduce or avoid freezing damage, and achieve the purpose of improving survival rate. The pre-culture medium is mainly a medium to which sucrose is added. The pre-cultivation method described in step (2) is to culture the adventitious buds in the MS liquid medium containing 0.3M sucrose in dark at 25° C. for 24 hours. Due to the high concentration of the plant vitrification solution, when the adventitious buds are transferred into the plant vitrification solution, the cells will be dehydrated quickly, which is easy to cause damage to the shoot tip. Therefore, the adventitious buds need to be loaded during vitrification protection. The pretreatment method described in step (3) is to treat the pre-cultured adventitious buds with a loading solution for 20 minutes. The composition of the transfer solution was MS basal medium supplemented with 2M glycerol and 0.4M sucrose. The plant vitrification solution described in step (4) is composed of PVS3 solution, which is MS basal medium supplemented with 50% (w/v) glycerol and 50% (w/v) sucrose. The plant vitrification solution processing time described in step (4), the round-leaved sage and the beautiful sage were treated with PVS3 for 40 minutes, and the S. PVS3 treatment for 80 minutes. The step (4) is as follows: the adventitious buds are transferred to a freezing tube containing a plant vitrification solution placed at room temperature, and each tube is equipped with 10 shoot tips. The step (5) is as follows: first transfer the 5 leaf discs to the aseptic aluminum foil strips, then directly put the aluminum foil strips into the cryopreservation tube placed in liquid nitrogen, and wait until the cooling process is completed Transfer the cryopreservation tubes to a liquid nitrogen tank for cryopreservation. The present invention also provides a method for reculturing Gesneriaceae plants after cryopreservation. Using the above method for cryogenic preservation, the leaf disks of five species of Gesneriaceae plants preserved at cryogenic temperatures are now thawed, and then cultured The plants of these five Gesneriaceae plants can be obtained. The thawing process is to take the aluminum foil strip out of the liquid nitrogen, quickly insert it into 10 ml of unloading solution (unloading solution) in a 6 cm diameter Petri dish, and treat it at 25° C. for 20 minutes. The composition of the unloading solution was MS basal medium supplemented with 1.2M sucrose. After the end of the unloading solution treatment, the leaf discs were transferred to recovery medium. The composition of recovery medium was MS basal medium supplemented with 0.09M sucrose, 0.5mg l ^-1 BA and 2.5gl ^-1 Phytagel. The thawed adventitious buds were first cultured in the dark at 25°C for 7 days, and when the adventitious buds turned green and showed signs of germination, they were transferred to MS solid medium, at 25°C, photoperiod 14/10 hours (light/dark), light intensity Incubate at 40 μmol m ^-2 s ^-1 .

Example 1:

Cryopreservation of five kinds of Gesneria:

Experimental materials: five kinds of plants of Gesneriaceae: rotundum oleifera, beautiful lipophyte, hanging stone, tooth leaf hanging stone and Baoshan hanging stone.

Pre-culture medium: liquid medium supplemented with 0.3M sucrose MS;

Loading solution: MS basal medium supplemented with 2M glycerol and 0.4M sucrose;

Plant vitrification solution PVS3: MS basal medium supplemented with 50% (w/v) glycerol and 50% (w/v) sucrose;

Recovery medium: MS basal medium supplemented with 0.09M sucrose, 0.5mg l ^-1 BA and 2.5gl ^-1 Phytagel (plant gel).

method:

Select the leaf discs of Lettuce rotundum that have completed the induction of adventitious buds, and pre-cultivate them in MS medium with a sucrose concentration of 0.3M in the dark for 24 hours, and treat the shoot tips in the loading solution at 25°C for 20 minutes, and then transfer to Plant vitrification solution PVS3 was treated at 25°C for 40 minutes. Then transfer the leaf disc to a sterile aluminum foil strip, insert the aluminum foil strip directly into the liquid nitrogen with a pair of fine tweezers, and when no more air bubbles are generated, transfer the aluminum foil strip to a cryotube placed in liquid nitrogen, and put it into Store in liquid nitrogen.

The leaf disks of Lettuce glabra which had been induced by adventitious buds were selected and pre-cultured in MS medium with a sucrose concentration of 0.3M in a dark environment for 24 hours, and the leaf disks were treated in the loading solution at 25°C for 20 minutes, and then transferred to Plant vitrification solution PVS3 was treated at 25 °C for 80 min. Then transfer the leaf disc to a sterile aluminum foil strip, insert the aluminum foil strip directly into the liquid nitrogen with a pair of fine tweezers, and when no more air bubbles are generated, transfer the aluminum foil strip to a cryotube placed in liquid nitrogen, and put it into Store in liquid nitrogen.

Select the leaf discs of P. japonica that have completed the induction of adventitious buds, pre-cultivate them in the dark environment of MS medium with a sucrose concentration of 0.3M for 24 hours, treat the shoot tips in the loading solution at 25°C for 20 minutes, and then transfer them to the plant Vitrification solution PVS3 at 25°C for 60 minutes. Then transfer the leaf disc to a sterile aluminum foil strip, insert the aluminum foil strip directly into the liquid nitrogen with a pair of fine tweezers, and when no more air bubbles are generated, transfer the aluminum foil strip to a cryotube placed in liquid nitrogen, and put it into Store in liquid nitrogen.

The leaf discs of P. serrata dentata leaves that had been induced by adventitious buds were selected, and pre-cultured in MS medium with a sucrose concentration of 0.3M in a dark environment for 24 hours, and the leaf discs were treated in the loading solution at 25°C for 20 minutes. Then transfer to plant vitrification solution PVS3 and treat at 25° C. for 40 minutes. Then transfer the leaf discs to sterile aluminum foil strips, insert the aluminum foil strips directly into liquid nitrogen with a pair of fine forceps, and transfer the aluminum foil strips to cryotubes placed in liquid nitrogen when no more air bubbles are produced. Take 5 adventitious buds and put them into liquid nitrogen for storage.

Select the leaf discs of Baoshan Lilywort that have been induced by adventitious buds, pre-cultivate them in MS medium with a sucrose concentration of 0.3M in the dark for 24 hours, treat the leaf discs in the loading solution at 25°C for 20 minutes, and then transfer To plant vitrification solution PVS3 at 25 ° C for 80 minutes. Then transfer the leaf disc to a sterile aluminum foil strip, insert the aluminum foil strip directly into the liquid nitrogen with a pair of fine tweezers, and when no more air bubbles are generated, transfer the aluminum foil strip to a cryotube placed in liquid nitrogen, and put it into Store in liquid nitrogen.

Recultivation and survival rate statistics:

When thawing, the aluminum foil strips were removed from the liquid nitrogen and quickly inserted into 10 ml of unloading solution in a 6 cm diameter Petri dish and treated at 25 °C for 20 min. After the end of the unloading solution treatment, the leaf discs were transferred to recovery medium. The thawed adventitious buds were first cultured in the dark at 25°C for 7 days, and when the adventitious buds turned green and showed signs of germination, they were transferred to MS solid medium, at 25°C, photoperiod 14/10 hours (light/dark), light intensity Incubate at 40 μmol m ^-2 s ^-1 .

The results of recovery culture showed that five species of Gesneriaceae plants recovered and grew well after cryopreservation and re-culture of adventitious buds (Fig. 1-5). The plant regeneration rates of the five species of Gesneriaceae, which are rotundum, beautiful lip, and genus, dentate and Baoshan genus, are 53.3%, 86.7%, and 70.0%, respectively. , 53.3%, 43.3% (Figure 8-12).

Example 2:

Cryopreservation of Litium rotundum—the effect of different plant vitrification solutions on the preservation effect:

Experimental material: the isolated shoot tip of Cystonia rotundum

Pre-culture medium: liquid medium of 0.3M sucrose MS;

Loading solution: MS basal medium supplemented with 2M glycerol and 0.4M sucrose;

Plant vitrification solution PVS3: MS basal medium supplemented with 50% (w/v) glycerol and 50% (w/v) sucrose; PVS2: MS basal medium supplemented with 15% (w/v) glycerol, 15% ethylene glycol , 15% dimethylsulfoxide and 0.4M sucrose.

Recovery medium: MS basal medium supplemented with 0.09M sucrose, 0.5mg l ^-1 BA and 2.5gl ^-1 Phytagel.

method:

Select the shoot tip of Litium rotundum, pre-cultivate it in MS medium with 0.3M sucrose in dark environment for 24 hours, treat it with loading solution at 25°C for 20 minutes, and then pass it through PVS2 and PVS3 respectively at 25°C Treatment for 20, 40 and 60 minutes. Then transfer the shoot tip to a sterile aluminum foil strip, insert the aluminum foil strip directly into the liquid nitrogen with a pair of fine forceps, and when there are no more air bubbles, transfer the aluminum foil strip to a cryotube placed in liquid nitrogen and keep Store in liquid nitrogen.

Recultivation and survival rate statistics:

When thawing, the aluminum foil strips were removed from the liquid nitrogen and quickly inserted into 10 ml of unloading solution in a 6 cm diameter Petri dish and treated at 25 °C for 20 min. After the end of the unloading solution treatment, the shoot tips were transferred to recovery medium. Thawed shoot tips were first cultured in the dark at 25°C for 7 days, and transferred to MS solid medium when the adventitious buds turned green and showed signs of germination. At 25°C, the photoperiod was 14/10 hours (light/dark), and the light intensity Incubate at 40 μmol m ^-2 s ^-1 .

The recovery culture results showed that: the regeneration rate of shoot tip of C. rotundumi cryopreserved under PVS2 treatment for 20-60 minutes was not more than 10%, and the experimental stability was relatively poor (Fig. 6). When PVS3 was treated for 20 minutes, the regeneration rate of the shoot tip of C. rotundum was 13%; when the PVS3 treatment time was extended to 40 minutes, the regeneration rate of the shoot tip of C. rotundum rose to 20%; when PVS3 When treated for 60 minutes, the shoot tip regeneration rate appeared to 10% (Fig. 7). Therefore, PVS3 is a more ideal plant vitrification solution in the cryopreservation of Gesneriaceae plants.

Example 3:

Cryopreservation of Litium rotundum—the effect of different explants on the effect of cryopreservation:

Experimental material: Limonaria rotundum. The leaves were cut to propagate test-tube plantlets, and adventitious buds induced by stem tips and leaf disks were obtained.

Pre-culture medium: liquid medium of 0.3M sucrose MS;

Loading solution: MS basal medium supplemented with 2M glycerol and 0.4M sucrose;

Plant vitrification solution PVS3: MS basal medium supplemented with 50% (w/v) glycerol and 50% (w/v) sucrose;

Recovery medium: MS basal medium supplemented with 0.09M sucrose, 0.5mg l ^-1 BA and 2.5gl ^-1 Phytagel (plant gel).

method:

Select the adventitious buds or shoot tips induced on the discs of the leaves of Lettuce rotundum as explants, pre-cultivate them in MS medium with a sucrose concentration of 0.3M sucrose in a dark environment for 24 hours, and treat them with loading solution at 25°C 20 minutes, and then treated with PVS3 at 25°C for 20, 40 and 60 minutes respectively. Then transfer the adventitious buds to a sterile aluminum foil strip, insert the aluminum foil strip directly into the liquid nitrogen with a pair of fine tweezers, and when no more air bubbles are generated, transfer the aluminum foil strip to a cryotube placed in liquid nitrogen and keep Store in liquid nitrogen.

Recultivation and survival rate statistics:

When thawing, the aluminum foil strips were removed from the liquid nitrogen and quickly inserted into 10 ml of unloading solution in a 6 cm diameter Petri dish and treated at 25 °C for 20 min. After the end of the unloading solution treatment, the leaf discs were transferred to recovery medium. The thawed adventitious buds were first cultured in the dark at 25°C for 7 days, and then transferred to MS solid medium at 25°C with a photoperiod of 14/10 hours (light/dark) and a light intensity of 40 μmol m ^-2 s ^{- 1} culture.

Recovery culture results showed:

When PVS3 was treated for 20 minutes, the regeneration rate of the shoot tip of C. rotundum was 13%; when the PVS3 treatment time was extended to 40 minutes, the shoot tip regeneration rate rose to 20%; when the PVS3 treatment time was further increased , the regeneration rate of its shoot tips decreased to 10% (Fig. 7). When the adventitious buds induced by leaf discs were used as explants, the regeneration rate of the leaf discs of Lettuce rotundum reached 43.3% when PVS3 was treated for 20 minutes; when PVS3 was treated for 40 minutes, the leaf discs The regeneration rate of the slices increased to 53.3%; when the PVS3 treatment was further extended to 60 minutes, the regeneration rate of the leaf discs decreased to 36.7% (Fig. 8).

Example 4:

Ultra-low temperature preservation of Limonaria rotundum—the effect of vitrification treatment time on the preservation effect:

The plant vitrification solution can dehydrate plant cells and enter the cells to vitrify the cells during the cooling process, effectively preserving the cell structure. However, since the concentration of the plant vitrification solution is too high, and the cryoprotectant has a toxic effect on the cells, it is necessary to strictly control the treatment time of the plant vitrification solution. Under the premise of ensuring a sufficiently high survival rate, the processing time should be shortened as much as possible.

Experimental material: Limonaria rotifera

Pre-culture medium: 0.3M sucrose MS liquid medium; loading solution: MS basal medium supplemented with 2M glycerol and 0.4M sucrose;

Plant vitrification solution PVS3: MS basal medium supplemented with 50% (w/v) glycerol and 50% (w/v) sucrose;

Recovery medium: MS basal medium supplemented with 0.09M sucrose, 0.5mg l ^-1 BA and 2.5gl ^-1 Phytagel.

method:

Select the leaf discs of Lettuce rotundum leaves that have been induced by adventitious buds, pre-cultivate them in MS medium with a sucrose concentration of 0.3M sucrose in the dark for 24 hours, treat them with the loading solution at 25°C for 20 minutes, and then pass them through PVS3 in the dark environment. 25°C for 20, 40 and 60 minutes, respectively. Then transfer the leaf disc to a sterile aluminum foil strip, insert the aluminum foil strip directly into the liquid nitrogen with a pair of fine forceps, and when no more air bubbles are generated, transfer the aluminum foil strip to a cryotube placed in liquid nitrogen, and Keep in liquid nitrogen storage.

Recultivation and survival rate statistics:

When thawing, the aluminum foil strips were removed from the liquid nitrogen and quickly inserted into 10 ml of unloading solution in a 6 cm diameter Petri dish and treated at 25 °C for 20 min. After the end of the unloading solution treatment, the leaf discs were transferred to recovery medium. The thawed adventitious buds were first cultured in dark at 25°C for 7 days, and then transferred to MS solid medium at the end of the dark culture. At 25°C, the photoperiod was 14/10 hours (light/dark), and the light intensity was 40 μmol m ^-2 s ^{- 1} culture.

Recovery culture results showed:

When PVS3 was treated for 20 minutes, the regeneration rate of the leaf discs of C. rotundum reached 43.3%; when the PVS3 treatment was extended to 40 minutes, the regeneration rate of the leaf discs rose to 53.3%; when the PVS3 treatment was further extended By the time of 60 minutes, the regeneration rate of its leaf discs had dropped to 36.7% (Fig. 8).

Example 5:

Ultra-low temperature preservation of Licoria chinensis—the effect of vitrification treatment time on the preservation effect:

Experimental material: beautiful lip column lettuce

Pre-culture medium: liquid medium of 0.3M sucrose MS;

Loading solution: MS basal medium supplemented with 2M glycerol and 0.4M sucrose;

Plant vitrification solution PVS3: MS basal medium supplemented with 50% (w/v) glycerol and 50% (w/v) sucrose;

Recovery medium: MS basal medium supplemented with 0.09M sucrose, 0.5mg l ^-1 BA and 2.5gl ^-1 Phytagel.

method:

Select the leaf discs of P. glabra that have completed the induction of adventitious buds, pre-cultivate them in MS medium with a sucrose concentration of 0.3M sucrose in the dark for 24 hours, treat them with the loading solution at 25°C for 20 minutes, and then pass them through PVS3 at 25°C. °C for 20, 40 and 60 minutes, respectively. Then transfer the adventitious buds to a sterile aluminum foil strip, insert the aluminum foil strip directly into liquid nitrogen with a pair of fine tweezers, and when no more air bubbles are produced, transfer the aluminum foil strip to a cryotube placed in liquid nitrogen and keep Store in liquid nitrogen.

Recultivation and survival rate statistics:

When thawing, the aluminum foil strips were removed from the liquid nitrogen and quickly inserted into 10 ml of unloading solution in a 6 cm diameter Petri dish and treated at 25 °C for 20 min. After the end of the unloading solution treatment, the leaf discs were transferred to recovery medium. The thawed adventitious buds were first cultured in dark at 25°C for 7 days, and then transferred to MS solid medium at the end of the dark culture. At 25°C, the photoperiod was 14/10 hours (light/dark), and the light intensity was 40 μmol m ^-2 s ^{- 1} culture.

Recovery culture results showed:

When PVS3 was treated for 20 minutes, the regeneration rate of cryopreserved leaf discs was 93.3%; when PVS3 was treated for 40 minutes, the regeneration rate of cryopreserved leaf discs was 86.7%; and when PVS3 was treated for 60 minutes, the regeneration rate of The rate was 100% (Fig. 9). Although there were differences between the cryopreservation regeneration rates of 20, 40, and 60 min PVS3 treatments, they were all very close to and reached 100%, showing that this protocol was very efficient for the beautiful L. chinensis. In the cryopreservation experiment with the goal of long-term storage of Licorice chinensis, the treatment time of PVS3 can be selected at any time point between 20-40 minutes, which is convenient for operation.

Embodiment 6:

Ultra-low temperature preservation of Lithospermia chinensis—the effect of vitrification treatment time on the preservation effect:

Experimental material: hanging stone lip column lettuce

Pre-culture medium: liquid medium of 0.3M sucrose MS;

Loading solution: MS basal medium supplemented with 2M glycerol and 0.4M sucrose;

Plant vitrification solution PVS3: MS basal medium supplemented with 50% (w/v) glycerol and 50% (w/v) sucrose;

Recovery medium: MS basal medium supplemented with 0.09M sucrose, 0.5mg l ^-1 BA and 2.5gl ^-1 Phytagel.

method:

Select the leaf discs of P. japonica that have completed the induction of adventitious buds, pre-cultivate them in the dark environment of MS medium with a sucrose concentration of 0.3M sucrose for 24 hours, treat them with the loading solution at 25°C for 20 minutes, and then pass them through PVS3 at 25°C. Treat for 40, 60 and 80 minutes respectively. Then transfer the adventitious buds to a sterile aluminum foil strip, insert the aluminum foil strip directly into the liquid nitrogen with a pair of fine tweezers, and when no more air bubbles are generated, transfer the aluminum foil strip to a cryotube placed in liquid nitrogen and keep Store in liquid nitrogen.

Recultivation and survival rate statistics:

When thawing, the aluminum foil strips were removed from the liquid nitrogen and quickly inserted into 10 ml of unloading solution in a 6 cm diameter Petri dish and treated at 25 °C for 20 min. After the end of the unloading solution treatment, the leaf discs were transferred to recovery medium. The thawed adventitious buds were first cultured in dark at 25°C for 7 days, and then transferred to MS solid medium at the end of the dark culture. At 25°C, the photoperiod was 14/10 hours (light/dark), and the light intensity was 40 μmol m ^-2 s ^{- 1} culture.

Recovery culture results showed:

When PVS3 was treated for 40 minutes, the regeneration rate of cryopreserved leaf discs was 33.3%; when PVS3 treatment was extended to 60 minutes, the regeneration rate of cryopreserved leaf discs increased significantly to 70.0%; when PVS3 treatment was further extended By 80 minutes, the regeneration rate had dropped to 15.0% (FIG. 10). The above results indicated that the leaves discs of S. japonicus were more sensitive to the treatment time of PVS3.

Embodiment 7:

Ultra-low temperature preservation of Lemonia dentifera: The effect of vitrification treatment time on the preservation effect:

Experimental material: Dendrophylla japonicus

Pre-culture medium: liquid medium of 0.3M sucrose MS;

Loading solution: MS basal medium supplemented with 2M glycerol and 0.4M sucrose;

Plant vitrification solution PVS3: MS basal medium supplemented with 50% (w/v) glycerol and 50% (w/v) sucrose;

Recovery medium: MS basal medium supplemented with 0.09M sucrose, 0.5mg l ^-1 BA and 2.5gl ^-1 Phytagel.

method:

The discs of P. serrata leaves that had been induced by adventitious buds were selected, pre-cultured in MS medium with a sucrose concentration of 0.3M sucrose in the dark for 24 hours, treated with loading solution at 25°C for 20 minutes, and then passed through PVS3 in the dark environment. 25°C for 60, 80 and 100 minutes, respectively. Then transfer the adventitious buds to a sterile aluminum foil strip, insert the aluminum foil strip directly into the liquid nitrogen with a pair of fine tweezers, and when no more air bubbles are generated, transfer the aluminum foil strip to a cryotube placed in liquid nitrogen and keep Store in liquid nitrogen.

Recultivation and survival rate statistics:

When thawing, the aluminum foil strips were removed from the liquid nitrogen and quickly inserted into 10 ml of unloading solution in a 6 cm diameter Petri dish and treated at 25 °C for 20 min. After the end of the unloading solution treatment, the leaf discs were transferred to recovery medium. The thawed adventitious buds were first cultured in dark at 25°C for 7 days, and then transferred to MS solid medium at the end of the dark culture. At 25°C, the photoperiod was 14/10 hours (light/dark), and the light intensity was 40 μmol m ^-2 s ^{- 1} culture.

Recovery culture results showed:

When PVS3 treatment was 60 minutes, the regeneration rate of cryopreserved leaf discs was 33.3%; when PVS3 treatment was extended to 80 minutes, the regeneration rate of cryopreserved leaf discs was 53.3%; when PVS3 treatment was further extended to 100 minutes , the regeneration rate decreased to 36.7% (Fig. 11).

Embodiment 8:

Ultra-low temperature preservation of Baoshan Diaoshi Limitophyllum: Influence of vitrification treatment time on preservation effect:

Experimental material: Baoshan hanging stone lip column lettuce moss

Pre-culture medium: liquid medium of 0.3M sucrose MS;

Loading solution: MS basal medium supplemented with 2M glycerol and 0.4M sucrose;

Plant vitrification solution PVS3: MS basal medium supplemented with 50% (w/v) glycerol and 50% (w/v) sucrose;

Recovery medium: MS basal medium supplemented with 0.09M sucrose, 0.5mg l ^-1 BA and 2.5gl ^-1 Phytagel.

method:

Select the leaf discs of Baoshan succulent leaves that have been induced by adventitious buds, pre-cultivate them in MS medium with a sucrose concentration of 0.3M sucrose in the dark for 24 hours, treat them with the loading solution at 25°C for 20 minutes, and then pass them through PVS3 at 25°C. °C for 60, 80 and 100 minutes, respectively. Then transfer the adventitious buds to a sterile aluminum foil strip, insert the aluminum foil strip directly into the liquid nitrogen with a pair of fine tweezers, and when no more air bubbles are generated, transfer the aluminum foil strip to a cryotube placed in liquid nitrogen and keep Store in liquid nitrogen.

Recultivation and survival rate statistics:

When thawing, the aluminum foil strips were removed from the liquid nitrogen and quickly inserted into 10 ml of unloading solution in a 6 cm diameter Petri dish and treated at 25 °C for 20 min. After the end of the unloading solution treatment, the leaf discs were transferred to recovery medium. The thawed adventitious buds were first cultured in dark at 25°C for 7 days, and then transferred to MS solid medium at the end of the dark culture. At 25°C, the photoperiod was 14/10 hours (light/dark), and the light intensity was 40 μmol m ^-2 s ^{- 1} culture.

Recovery culture results showed:

When PVS3 was treated for 60 minutes, the regeneration rate of cryopreserved leaf disks was 26.7%; when PVS3 treatment was extended to 80 minutes, the regeneration rate of cryopreserved leaf disks was 43.3%; when PVS3 treatment was further extended to 100 minutes , the regeneration rate was 43.3% (Fig. 12).

Claims (10)

1. Gesneriaceae cryopreservation method, is characterized in that the method comprises the steps:

(1) get Gesneriaceae leaf disk under aseptic condition, leaf disk is proceeded in adventitious bud induction culture base and complete adventitious bud inducing;

(2) preculture is carried out to the leaf disk completing adventitious bud inducing;

(3) get the leaf disk of step (2), proceed in the cryovial being equipped with and loading liquid, at 25 DEG C, load process 20 minutes;

(4) leaf disk in step (3) is placed in plant vitrification solution, freezing processing;

(5) step (4) process after leaf disk drop in liquid nitrogen and preserve.

2. store method according to claim 1, it is characterized in that, the Gesneriaceae of described step (1) is Gesneriaceae lip post lettuce tongue, beautiful lip post lettuce tongue, Lysionotus carnosus Hemsl. (Lysionotus pauciflorus Maxim.), tingia Lysionotus carnosus Hemsl. (Lysionotus pauciflorus Maxim.), Lysionotus sulphureoides, described adventitious bud inducing is the leaf disk that the plantlet in vitro blade card punch getting above-mentioned plant breaks into 3mm, carries out adventitious bud inducing.

3. store method according to claim 1, is characterized in that, the preculture of described step (2) to complete the leaf disk of adventitious bud inducing in the MS liquid nutrient medium of the sucrose containing 0.3M, light culture 24 hours at 25 DEG C.

4. store method according to claim 1, is characterized in that, the loading liquid described in described step (3) is that MS basal medium adds 2M glycerine and 0.4M sucrose.

5. store method according to claim 1, is characterized in that, the plant vitrification solution in described step (4) is PVS3 solution, adds 50% (w/v) glycerine and 50% (w/v) sucrose by MS basal medium.

6. store method according to claim 1, it is characterized in that, the plant vitrification solution processing time in described step (4) is: roundleaf lip post lettuce tongue, beautiful lip post lettuce tongue, Lysionotus carnosus Hemsl. (Lysionotus pauciflorus Maxim.), tingia Lysionotus carnosus Hemsl. (Lysionotus pauciflorus Maxim.), Lysionotus sulphureoides are respectively through PVS3 process 40,40,60,80,80 minutes.

7. store method according to claim 1, is characterized in that, described step (3) is equipped with in plant vitrification solution cryovial at leaf disk is transferred to 25 DEG C, and often pipe fills 10 leaf disks.

8. store method according to claim 1, is characterized in that, described step (4) freezing processing is first transferred to by leaf disk on sterile aluminum foil bar, then described aluminum foil strip directly inserted liquid nitrogen.

9. according to claim 1 ?store method described in 8 any one, it is characterized in that, Gesneriaceae after step (5) is preserved is cultivated again, the leaf disk of the Gesneriaceae of Excised Embryos is thawed, again through renewal cultivation, described thaws for being taken out from liquid nitrogen by aluminum foil strip, and quick insertion 10ml MS basal medium adds in the unloading solution of 1.2M sucrose, processes 20 minutes at 25 DEG C.

10. store method according to claim 9, is characterized in that, described renewal cultivation the leaf disk after thawing is proceeded to MS basal medium add 0.09M sucrose, 0.5mg l ^?1bA and 2.5gl ^?1cultivate in the recovery media of Phytagel, first light culture 7 days at 25 DEG C, after at 25 DEG C, photoperiod illumination/dark 14/10 hour, light intensity 40 μm of ol m ^?2s ^?1lower cultivation.